Freeze Drying of Cell-Containing Regenerative Medicine Products to Enable Long Term Storage: Initial Studies Using Mammalian Red Blood Cells

1. Freeze-drying of cell-containing
regenerative medicine products
to enable long term storage
Initial studies using mammalian red blood cells
Dr Kevin R. Ward B.Sc. Ph.D. MRSC
Director of Research & Development
BTL, Winchester SO23 0LD, UK
Tel: +44 (0)1962 841092
E-mail: kward@biopharma.co.uk

2. Synopsis of Presentation
• Reasons for looking at blood / red blood
cells
• Aims of study
• Issues with freeze-drying of RBC
• Variables studied for human RBC
• Results 1 – RBC ‘survival’
• Results 2 – Haemoglobin oxidation
• Next steps

3. Why Blood?
• In the UK, large volumes of donated blood are
discarded due to stability issues
• NHSBT assigns donated blood a shelf life of 35
days at 2-8°C
• Stocks of blood for transfusion rarely exceed
enough for more than 1 week
• Therefore there is considerable value in
achieving more stable material
• Freeze-drying offers the opportunity of
stabilising whole blood / blood components

4. Why Red Blood Cells (RBC)?
• RBC present a challenge – other factors
have successfully been lyophilised but no
reports of 100% success with RBC for
either freeze-thawing or freeze-drying
• RBC membrane not as robust as
nucleated cells – therefore, more cryo- /
lyo- protection is likely to be needed
• RBC lysis is relatively simple to quantify in
terms of haemoglobin leakage

5. Aims of Study
• To investigate a series of variables
(formulation and process) on the survival of
RBC after freeze-drying + rehydration
• To quantify RBC ‘survival’ by haemoglobin
release in supernatant compared with pellet
• To quantify level of haemoglobin oxidation
by UV-visible spectrophotometry
(comparing Hb, met-Hb, oxy-Hb)

6. Issues with RBC lyophilisation
• Freezing damage:
– from ice crystal growth
– from pH gradients
– from freeze-concentration / osmotic effects
• Drying damage:
– Physical action of ice removal on membrane
– Dehydration causing deformation of RBC
• Rehydration damage:
– Concentration and pH effects (e.g. localised
hypotonic / acidic / alkaline regions, causing lysis)
– Wetting issues exacerbating the above effects

7. Variables for Human RBC Study
Variable: Option 1 Option 2 Option 3
Buffer type Buffer 1 Buffer 2 Buffer 3
Buffer concentration High Medium Low
Protectant type Non-polymer Non- Polymer
1 polymer 2
Protectant concentration High Medium Low
Cooling rate High Medium Low
1°D shelf temperature High Medium Low
Rehydration solution Salt Buffer Polymer

8. Additionally…
• Our collaborating academic partner had shown
that a novel biopolymer had the ability of
providing a mechanism of intracellular loading of
protectant
• Therefore, a duplicate set of samples would also
be exposed to polymer
• BUT 2 x 37 sets of conditions plus controls would
be ~4,400 samples… and in triplicate, this would
be 13,200 samples… and 3 UV/vis cuvettes
from each sample would give 39,600 cuvettes!
• The project timeframe did not allow for a DoE
approach. Therefore, the study was rationalised
and only selected combinations were studied.

9. RESULTS (1) – RBC ‘survival’
• Levels of RBC survival of 96% were achieved for the combination below
• Surprisingly, this was achieved in the absence of biopolymer, which
implies that intracellular protectant may not be necessary
• Oxidation level of Hb was quite high (60%)
Variable: Option 1 Option 2 Option 3
Buffer type Buffer 1 Buffer 2 Buffer 3
Buffer concentration High Medium Low
Protectant type Non- Non- Polymer
polymer 1 polymer 2
Protectant concentration High Medium Low
Cooling rate High Medium Low
1°D shelf temperature High Medium Low
Rehydration solution Salt Buffer Polymer

10. RESULTS (2) – Hb oxidation
• While the biopolymer did not necessarily lead to
higher RBC survival under the conditions employed
here, it was noted that it was only in samples
containing the biopolymer that the haemoglobin
oxidation was reduced to below detectible levels,
and was typically below 10%
• Prevention of Hb oxidation may have been
attributable to the direct action of the polymer itself
and/or to the presence of intracellular protectant
facilitated by the presence of the polymer

11. What Next?
• Further studies on RBC, building on the data from
this study:
– Using a DoE approach to cover all combinations of
variables identified here
– Extending the number of formulation and processing
variables
– Looking in detail at a novel method of iso-osmotic RBC
concentration / rehydration using specialist membranes
– Examining RBC deformability upon rehydration
– Fine tuning polymer use to match best non-polymer
survival rate
– Looking at long term stability in the freeze-dried state
• Application of the principles of this study to the
freeze-drying of nucleated cells

12. SUMMARY
• A large number of combinations of formulation
and process variables were tested in the freeze-
drying of RBC
• Best RBC survival rate was 96%, but with 60%
Hb oxidation
• Use of novel biopolymer led to Hb oxidation
levels below detectable limits, but maximum
survival was 85%
• Valuable lessons learned that will be applied to
further studies on RBC and nucleated cells

13. Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK
Tel: +44 (0)1962 841092 Web: www.btl-solutions.net